The current resurgence of antibiotic-resistant organisms underscores the importance of gaining a better understanding of antibiotic mechanisms, resistance modes and the structural features necessary for optimal effectiveness. The overall objective of this proposal is to learn how five structurally different antibiotics inhibit the process of bacterial cell growth. This investigation will explore the new observation that macrolide antibiotics as well as the ketolides, lincosamides, streptogramin B compounds and oxazolidinones can all inhibit the assembly of the large ribosomal subunit in bacterial cells. Ribosome formation will be analyzed in Staphylococcus aureus and Escherichia coli cells to define the inhibitory features of these compounds. The mechanism of subunit assembly inhibition will be tested by examining the components of the subunit precursor particles which accumulate in the presence of the antibiotic. Aspects of the breakdown of the inhibited assembly intermediate will also be studied. Ribosomal subunits will be reconstituted from component RNAs and proteins to define the molecules involved as targets for assembly inhibition. An investigation of this assembly-sensitive site and the mode of inhibition of assembly will reveal how certain antibiotics can have two inhibitory activities. The findings from this work will help in assessing the effectiveness of existing antibiotics and in developing new compounds as antimicrobial agents.